Changes in DNA methylation patterns play an important role in tumorigenesis. The DNA methyltransferase 1 (DNMT1) protein represents a major DNA methyltransferase activity in human cells and is therefore a prominent target for experimental cancer therapies. However, there are only few available inhibitors and their high toxicity and low specificity have so far precluded their broad use in chemotherapy. Based on the strong conservation of catalytic DNA methyltransferase domains we have used a homology modeling approach to determine the three-dimensional structure of the DNMT1 catalytic domain. Our results suggest an overall structural conservation with other DNA methyltransferases but also indicate local conformational differences. To prove the validity of our model we used it as a template to design a novel derivative of the known DNA methyltransferase inhibitor 5-azacytidine. The resulting compound (N4-fluoroacetyl-5-azacytidine) functioned as an efficient inhibitor of DNA methylation in human tumor cell lines and also provides novel opportunities for pharmacological applications. (C) 2003 Elsevier Science (USA). All rights reserved.